Synthesis, biological evaluation and in-silico ADME studies of novel series of thiazolidin-2,4-dione derivatives as antimicrobial, antioxidant and anticancer agents

Background A novel series of thiazolidine-2,4-dione molecules was derived and their chemical structures were established using physiochemical parameters and spectral techniques (1H-NMR, IR, MS etc.). The synthesized molecule were then evaluated for their antioxidant, anticancer and antimicrobial potential. Results and discussion Serial tube dilution method was employed to evaluate the antimicrobial potential against selected fungal and bacterial strains by taking fluconazole and cefadroxil as reference antifungal and antibacterial drugs respectively. 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity was used to assess the antioxidant potential of the synthesized analogues. Further, the anticancer potential of the selected molecules was assessed against DU-145 cancer cell lines using MTT assay. The drug-likeness was also evaluated by studying in-silico ADME parameters of the synthesized analogues. Conclusion In antioxidant evaluation studies, the analogue H5 with IC50 = 14.85 μg/mL was found to be the most active molecule. The antimicrobial evaluation outcomes suggested that the molecules H5, H13, H15 and H18 possessed moderate to promising activity against the selected species of microbial strains having MIC range 7.3 µM to 26.3 µM. The results of anticancer evaluation revealed that all the screened derivatives possess mild anticancer potential. The in-silico ADME studies revealed that all the compounds were found to be drug-like.


Introduction
The World Health Organization (WHO) reports indicate that the microbial drug resistance (MDR) caused due to continuous use of presently available antibiotics and the development of resistance against presently available anticancer drugs is the major concern for human life worldwide now a days [1,2]. The clinical effectiveness of currently using antibiotics against most of the MDR strains, viz. vancomycin-resistant enterococci (VRE), multidrug-resistant staphylococcus aureus (MRSA) etc. is shrinking constantly [3,4]. This prompts the medicinal chemist/pharmacologist to explore their research to find the alternative antimicrobial drug therapies [5].
Cancer, is one of the most terrible diseases and is the leading cause of death worldwide which accounts for approximately 17% of the total causalities. It has been charterized by uncontrolled and abnormal cell growth [6]. Inhibition of DNA replication and transcription to restrain the growth of tumor cells by currently used chemotherapeutic drugs is highly toxic [7], hence there is no ideal therapy currently available to cure cancer. This prompts medicinal chemists and researchers to find newer compounds having good antimicrobial and anticancer potential [2] with an innovative mode of action and lesser toxic effects.
Due to extreme surge in the cost of discovering new drug candidates, the drug discovery strategy has shifted to assess the comprehensive drug properties of the molecules under study at the earliest, for the clinical success of the drug candidates [29]. Pharmacokinetic parameters like absorption, distribution, metabolism and excretion (ADME) play a vital role in dose defining, overall safety margins and dose intervals during the drug development process [30]. Optimization of these parameters for a new chemical moiety having specific biological potential to be orally active is extremely important [5]. The molecules with poor ADME parameters may show unexpected toxicity, leading to withdrawal from the market and hence causing large financial losses [31].
Based on the literature survey and in continuation with our previous research efforts; in the present study, some new (4-oxothiazolidin-2-yl)thiazolidine-2,4-dione derivatives were synthesized and screened for antimicrobial, anticancer and antioxidant potential along with in-silico evaluation of ADME parameters of synthesized molecules.

Results and discussion
Chemistry TZD derivatives (H1-H19) were synthesized by employing a synthetic procedure as shown in Scheme 1. Initially, 2-chloroacetic acid was allowed to react with thiourea in presence of concentrated HCl to get TZD (INT-I).

Antimicrobial screening
The in vitro antimicrobial evaluation of the synthesized analogues was carried out using a serial tube dilution procedure [32] (Table 2   possess superior activity against both the selected strains of fungus i.e., A. niger and C. albicans while antibacterial screening results exhibited mild to moderate activity against the selected strains in comparison to cefadroxil as standard drug. So, these molecules can be viewed as lead structures for further development and optimization into potent antimicrobial agents.

Antioxidant evaluation
DPPH free radical scavenging assay was performed to assess the antioxidant potential of the newly synthesized derivatives using ascorbic acid as a reference drug [33]. DPPH assay is among the most utilized methods used for assessing the antioxidant potential of a compound which is based on a chain-breaking mechanism. DPPH is a stable free radical which can be transformed into a constant diamagnetic molecule by accepting hydrogen or an electron radical from the antioxidant compound [34]. The DPPH solution (methanolic) exhibit a strong absorption band at 517 nm. As DPPH radical reacts with the antioxidant/ reducing agent, a new bond is generated which leads to decreases in the color intensity of the solution. As the strength of antioxidants in the solution is increased, the DPPH radical takes up a greater number of electrons, leading to a loss in the color intensity of the solution from purple to pale yellow which is monitored spectrophotometrically at 517 nm [35]. The IC 50 value (μg/mL) and % inhibition for all the synthesized molecules were calculated. The antioxidant screening assay revealed that the derived molecules were more potent than the reference drug itself. Further, the antioxidant screening showed compound H5 (IC 50 = 14.85 μg/mL) be the most potent. Antioxidant evaluation results are depicted in Table 3 and Fig. 4.

ADME results
The synthesized derivatives (H1-H19) were submitted to QikProp module of Schrödinger software 2020-4 (Maestro version 12.5) for the calculation of ADME parameters [36]. The results of ADME studies were   Table 4.

Anticancer potential
Anticancer potential of three synthesized derivatives viz. H2, H10 and H11 were tested for their in vitro anticancer potential against prostate cancer cell line (DU-145) using MTT assay. The results of anticancer evaluation of all the screened derivatives were summarized in Fig. 5.
• Presence of electron donating group (-OCH 3 ) at ortho position in molecule H15 enhanced antibacterial activity against E. coli. • Substitution of nitro (-NO 2 ) group at meta position (electron withdrawing) in molecule H13 enhanced antifungal activity against A. niger. • Presence of (-F) group (electron withdrawing) at para position in molecule H18 enhanced the antibacterial activity against S. typhi and B. subtilis whereas substitution of aliphatic group dodecyl in the derived molecule H5, enhanced the antioxidant activity and also exhibited better antimicrobial potential against S. aureus and C. albicans.

Conclusion
A series of thiazolidin-2,4-dione clubbed with thiazolidin-4-one molecules was synthesized and then screened for its antimicrobial, anticancer and antioxidant potential. The molecules H5, H13, H15 and H18 exhibited In the antioxidant evaluation assay, the compound H5 was found to be the best antioxidant molecule among the synthesized series with an IC 50 value of 14.85 μg/mL. ADME studies revealed that all the compounds found to be drug-like to be orally active as all the parameters of the compounds was found within Lipinski's rule of five. These derivatives can be used as lead structures for further modification/optimization into more potent antimicrobial, anticancer and antioxidant drug molecules with least toxicity.

Experimental
The chemicals used in the synthetic work were either of AR or LR grade purchased from different vendors and were used as such without any further purification. The melting point (m.p.) of the synthesized compounds was determined by using Stuart scientific SMP3  Step 1: Synthesis of thiazolidin-2,4-dione TZD (INT-I):Aqueous solution of thiourea (0.06 mol in 15 mL of water) was added dropwise to the aqueous solution of chloroacetic acid (0.06 mol in 15 mL of water) and stirred constantly till the formation of white precipitates. 6 mL of concentrated HCl was then added dropwise in the above mixture and then refluxing was carried for 10 h. Fine needle shaped crystals of TZD (INT-I) were formed on cooling which were filtered, dried and recrystallized from methanol [27].

In vitro antimicrobial evaluation
The synthesized molecules were screened for their antimicrobial potential employing serial tube dilution method [32] by comparing with marketed antibiotics; cefadroxil (antibacterial screening) fluconazole (antifungal screening). The evaluation was carried out using both  [39] nutrient media. Dimethyl sulfoxide was used as diluting medium for the preparation of stock solutions of the reference and test molecules along with a control set of the same dilutions. Incubation of the samples at 37 ± 1 °C (24 h) for bacteria, at 37 ± 1 °C (48 h) for C. albicans and at 25 ± 1 °C (7 days) for A. niger, respectively. Results were recorded as MIC for the tested molecules that exhibited no observable growth of microorganisms in the test tube at the lowest possible concentration.

In vitro antioxidant assay
The antioxidant potential of synthesized thiazolidine-2,4-dione molecules was analyzed using DPPH free radical scavenging assay [40]. Synthesized compounds were diluted to 25 μg/mL, 50 μg/mL, 75 μg/mL and 100 μg/ mL concentration with methanol and kept in different test tube. To these test tubes equal quantity of 0.0039% DPPH in methanol was added followed by vigorous shaking. The test tubes containing the above mixture were then wrapped with silver foil paper and kept in a dark room for 30 min. Finally, a UV-Visible double beam spectrophotometer was used to measure the absorbance of the mixtures at 517 nm. The mean IC 50 value of at least three observations is presented in the data.

In vitro anticancer screening
The anticancer screening was done using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Firstly, cells of DU-145 were seeded (5 × 10 3 cells/well) at clear 96-well plates. After 24 h, synthesized derivatives in a concentration of 1 µM, 3 µM, 10 µM, 30 µM, 90 µM, 150 µM, 270 µM, 350 µM, 500 µM, 700 µM and 1 mM were added to each well to expose the cell lines for further 24 h. After the treatment, the 0.5 mg/mL MTT reagent solution was added and the plates were incubated at 37 °C for 4 h. MTT solution was then removed by inverting the well plate and 150 μL of DMSO was then added to dissolve insoluble formazan crystals. The optical density at 570 nm was determined spectrophotometrically. The anticancer potential was expressed as the relative cell viability (%) relative to the untreated control cells [41].